The variable complexity of peptide mixtures and occasional drifts lead to substantial variations in m/z and RT dimensions. Thus, label-free differential protein expression studies by LC-MS technology require alignment with respect to both RT and m/z to ensure that same proteins/peptides are compared from multiple runs.In proteomics studies, liquid chromatography coupled to mass spectrometry (LC-MS) has proven to be a powerful technology to investigate differential expression of proteins/peptides that are characterized by their peak intensities, mass-to-charge ratio .In this study, we propose a new strategy to align LC-MALDI-TOF data by combining quality threshold cluster analysis and support vector regression. Our method performs alignment on the basis of measurements in three dimensions (RT and m/z dimensions reveals that the use of intensity measurements enhances alignment performance.We demonstrate the suitability of our proposed method for alignment of LC-MALDI-TOF data through a previously published spike-in dataset and a new in-house generated spike-in dataset. A comparison of our method with other methods that utilize only  Proteomics has been extensively used to study the protein expression of cells under various physiological conditions. As an indispensable tool for proteomics, mass spectrometry (MS) can identify and determine the abundance of various proteins or peptides in easily accessible, non-invasive body fluid samples such as serum and urine. However, the presence of a large number of peptides or proteins and their dynamic range in the body fluid raise many challenges in the data analysis. A separation procedure prior to MS analysis has been utilized to reduce the complexity of samples. Liquid chromatography (LC) coupled with MS (LC-MS) has become a common platform in proteomics studies, since it combines the physical separation capabilities of LC with the mass analysis capabilities of MS of protein mixtures.RT) and mass-to-charge ratio (m/z), and quantifies them with peak intensity . Comparison of multiple LC-MS runs generated from different conditions  provides a comprehensive quantitative overview of thousands of peptide concentration among samples to identify differentially abundant peptides. One of the most significant challenges in label-free comparison of multiple LC-MS runs is to ensure that the peptide peaks are aligned correctly prior to identification of differential abundance. Without alignment, the same peptide may have different m/z and RT values across multiple runs due to m/z and RT drifts. The shifts in the m/z dimension typically have small deviation, but the RT dimension is prone to large and non-linear shifts due to the LC instrument conditions, peptide-peptide interactions, and peptide/protein modifications. Improper correction of m/z and RT shifts may lead to false discovery of biomarker candidates. Therefore, a reliable peak alignment strategy is required prior to comparing biological groups represented by multiple LC-MS runs.LC-MS characterizes peptides and proteins by their retention time  ,2, dynameviation . Moreovem/z dimension. Once clustering was completed, the dendrogram was cut into clusters, and all peaks belonging to a cluster were considered to be the same peak. A “consensus” peak was extracted by taking the mean m/z value of each cluster. Jaitly et al. [RT deviation for each run with regression methods. Lange et al. [Various peak-based alignment methods have been reported in the literature -7. In thy et al.  utilizede et al.  describeMost clustering methods typically involve the following steps in peak-based spectral alignment: (1) identify “high intensity” peaks; (2) cluster these peaks; (3) align the peaks with respect to the cluster center; and (4) use regression or other mathematical model to estimate shifts of all peaks and to correct the shifts.m/z and RT dimensions, ignoring peak intensity measurements that might be important to accurately align LC-MS runs. In addition, it is important to find a suitable threshold to cut the dendrogram tree or decide the number of clusters. Accurate estimation of the number of clusters is desired, but it is clearly a challenging task.There are some drawbacks in using the above-mentioned clustering-based approach for LC-MS alignment. For instance, most clustering methods only consider m/z, RT, and ion intensity measurements to perform clustering. Specifically, we use a partition-based clustering approach to find clusters by evaluating the quality of clusters and a non-linear regression to correct RT shifts. We evaluate the performance of this method through two spike-in datasets, an in-house generated dataset and a previously published dataset.In this study, we propose a new LC-MS alignment method based on cluster analysis of peaks. We define a distance metrics that involves This dataset consists of 12 LC-MALDI-TOF runs derived from two groups of samples: (1) serum samples without spike-in peptides, and (2) serum samples spiked with a mixture of peptides containing 40 fmol/μl of angiotensin (1296.70 Da), 26 fmol/μl of Glu1-Fibrinopeptide B (1570.70 Da), 40 fmol/μl of ACTH 1-17 fragment (2093.10 Da), and 30 fmol/μl of ACTH 18–39 fragment (2465.19 Da). Each group is comprised of three experimental replicates with each replicate measured in duplicate.Blood sample of a healthy volunteer was collected by a trained phlebotomist, and was processed to enrich native peptides in the low molecular weight fraction of serum. Forty-five microlitre of serum sample was desalted on C8 magnetic beads and ultrafiltered in 25% acetonitrile on 50 kDa Microcon membranes.Peptide separation was performed on a Tempo nano MDLC system  in conjunction with a Probot MALDI spotting device (Dionex-LC Packings). We concentrated 5μl of sample on a 0.5 cm x 300 μm i.d. C18 trap column using mobile phase A  delivered at 20 μl/min. The sample was then eluted onto a 150 mm x 75 μm i.d Vydac C18 column  using a linear gradient of solvent B  in solvent A as follows: from 5% to 60% of B in 180 min, switched to 95% solvent B for 10 min, followed by 20 min re-equilibration with solvent A at a constant flow rate of 0.3 μl/min. The elution of the column was mixed with a solution of 3.75 mg/ml α-cyano-4-hydroxycinnamic acid MALDI matrix (α-CHCA)  at the exit. The CHCA was prepared daily and delivered at a flow rate of 0.6 μl/min. The mixture of elution and CHCA was spotted into the MALDI plate through the Probot spotting system. Each spot represented a one-minute "fraction" (0.9 μl) of the reverse phase gradient. Triplicate runs of the sample, each containing 192 spots, were distributed on the MALDI plates.The spots representing chromatographic fractions were analyzed using ABI 4800 MALDI-TOF/TOF, equipped with a neodymium: yttrium-aluminum-garnet laser emitting at λ = 355 nm with a repetition rate of 200 Hz. An average of 50 shots at each of 20 positions was collected for a total of 1000 shots/spot in the reflector positive mode in the mass range of 800-5000 Da. Each MALDI plate was measured in duplicate.This dataset consists of 24 LC-MALDI-TOF runs, which were obtained from urine samples of three healthy volunteers. Aliquots of each sample were spiked with three peptides at four different concentrations , where 1X = 120 fmol/μl of human angiotensin II (1046.54 Da), 80 fmol/μl of neurotensin (1672.91 Da), 100 fmol/μl of ACTH 18–39 fragment (2465.19 Da). Each spike-in sample was measured in duplicate by off-line coupling of Ultimate 3000 nano-HPLC system and ABI 4800 MALDI-TOF/TOF. Each LC-MALDI-TOF run contained 358 fractions. Each fraction represented a 12-second interval (containing 15 μl of urine elution and 45 μl of α-CHCA matrix) of the LC gradient.m/z below 1000 were excluded. We performed binning with a bin size of 200 ppm, baseline correction by using the loess method, normalization by dividing each spectrum by its total ion count, denoising using wavelet shrinkage, and peak detection by finding local maxima of the denoised data. These preprocessing steps were applied at MALDI-TOF spectrum level. Furthermore, the peak intensities for each LC-MALDI-TOF run were normalized to the median intensity of one reference MALDI-TOF spectrum, which has the highest median intensity compared to the rest of spectra. The normalized peaks from all runs were combined together in three dimensions .In both datasets, all ions with Previously applied to identify coexpressed genes from microarray data, quality threshold (QT) clustering method was originally described by Heyer et al . It is bPrior to the QT cluster analysis, for each LC-MALDI-TOF run, we selected the peak candidates with intensity higher than the mean intensity of that specific run. The candidates from all runs were combined together to form a peaklist, and the peaklist was used in cluster analysis procedure. The QT clustering is an iterative algorithm and starts with a global set that includes all elements (peaks) of the dataset (peaklist), and returns a set of clusters that satisfies the quality threshold. Such threshold is defined in terms of cluster diameter. The algorithm includes the following steps:a) For each element, a candidate cluster seeded by this element is formed.b) Such cluster is iteratively added by element, which can minimize the increase in cluster diameter. This process continues until no element can be added without surpassing the diameter threshold.c) Once a cluster is generated, all elements within this cluster are excluded from the subsequent analysis.d) Repeat Steps a–c for the subsequent cluster identification, until all elements belong to a particular cluster.e) Clusters with fewer elements than a specific number are removed, and the elements (peaks) in these clusters are excluded. Other clusters are retained as qualified clusters.f) Iterate Steps a-e multiple times, each time stating from different candidate cluster seeds of the first element. The summation of the distances within the qualified clusters is used to compare different iterations. The iteration with the smallest summation of within-clusters is our solution and the qualified cluster information in this iteration is used in the regression analysis.1 and peak2  in the cluster analysis is a weighted Euclidean distance in the m/z, RT and intensity (int) dimensions, i.e., mzw, RTw, intw are weights associated with the three dimensions.The distance between peakRT, m/z, and log transformed intensity were standardized to have zero mean and unit standard deviation. RTw was set to one. Considering the measurement precision range of mass spectrometry and HPLC, the penalty for the m/z change should be much higher than the penalty for the RT change. Therefore, mzw was screened between 100 to 2000. In most cases, the penalty for the m/z change should be higher than the penalty for the intensity change. We screened intw between 0.1 to mzw/2. The minimum number of peaks within one cluster was set to two. The maximum cluster diameter was optimized along with the optimization of weight values  of the distance metric. A criterion used to screen the parameters was the ratio of the number of “good-behaved” clusters to the total number of clusters. The behavior of a cluster is defined as the ratio of the number of all runs to the number of replicate runs within this cluster.The RT deviation and to correct the shifts of all detected peaks. SVR maps the data into a higher-dimensional feature space via a non-linear kernel function, and a linear regression is conducted in this feature space [1x, 1y),….}, where x represents T and y denotes the average RT values with clusters, the SVR model f will be given byFollowing clustering, for each LC-MALDI-TOF run, a support vector regression (SVR) was applied to model re space . Let theK is a kernel function denoting a set of transformation , and b is the “bias”. SVR performs regression in the high-dimension feature space using ε-insensitive loss, and simultaneously reduce the model complexity by minimizing the Euclidean norm ||w||. This problem can be written as a convex optimization problem by minimizing 0.5||w|| subject toWhere This optimization problem can be transformed into the dual problem and its solution given byb are determined by maximizing the following Lagrangian expressionThe coefficients α, α*, and under the following conditions ofm/z < 1000 Da were excluded since matrix had negative impacts on signal peaks with mass below 1000. The alignment procedure was performed on 12 runs, 6 runs in each group. Table m/z ranging from 1000 to 5000. For samples without spike-in peptides, the average CV was decreased from 28% to 22%, and the average Pearson correlation coefficient was increased from 0.65 to 0.94. For samples with spike-in peptides, the average CV was decreased from 40% to 22%, and the average Pearson correlation coefficient was increased from 0.79 to 0.95. Fig. m/z and RT values of the four spike-in peptides before and after alignment.We evaluated our alignment algorithm with our in-house LC-MALDI-TOF dataset, which contained two groups of samples, 1) serum samples without spike-in peptides and 2) serum samples spiked with four peptides. All signals with  serum sa serum saRT shift before alignment. This RT shift could mask the relative peptides which introduce false biomarker discovery. As shown in Table We performed additional evaluation of our algorithm using the Benkali’s dataset , which rRT shifts in LC-MALDI-TOF data. Currently, the alignment of liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) runs is well discussed in the literature [RT shifts in LC-MALDI-TOF maps, a discrete alignment method is expected to be more efficient than continuous methods, which have been developed and extensively investigated in LC-ESI-MS data [We introduce a new method that utilizes the clustering concept to correct terature ,12-14. Hterature . To adju-MS data . Althoug-MS data ,15. Seco-MS data . This ism/z, RT, and intensity. Support vector regression is used to model and correct the RT shifts in the entire m/z and RT dimensions. Cluster analysis on the basis of peaks from LC-MALDI-TOF data allows us to determine clusters of peaks that potentially represent the same biological entities. Clustering method is especially suitable for alignment of LC-MALDI-TOF runs due to their high resolution and discrete property that allows us to easily breakdown the data into “peak” and “non-peak” regions compared with LC-ESI-MS runs.Taking the differences between LC-ESI-MS and LC-MADLI-TOF MS into consideration, in this paper we introduce a new peak alignment method for LC-MALDI-TOF data analysis. Our method uses QT cluster analysis to cluster the peaks based on Our method is evaluated through two datasets. The first dataset (in-house dataset) is generated by our group, containing experimental replicates. The CVs within specific groups are decreased from 28%-40% to 22%, and the correlation coefficients are increased from 0.65-0.79 to 0.94-0.95. The second dataset  is obtained from published literature containing biological replicated. The CVs within specific groups are decreased from 27%-33% to 15%-25%, and the correlation coefficients are increased from 0.31-0.50 to 0.89-0.93.RT, m/z, intensity) with those obtained from analysis of two dimensions (RT and m/z), only. In the in-house dataset, we observed that the use of three dimensions leads to significantly better correlation among samples that belong to the same group  were involved in those methods. The results are shown in Table mz, and wint), which increase the computational time. Future work will focus on addressing this limitation through optimization of the algorithm.As an example for comparison, Podwojski’s methods  were appNo conflict interest is declaredZT: conception and conduct of experimental design, development of data analysis tools, writing and revision of manuscript, figure


The best strategy to protect individuals against meningococcal disease is to immunize against multiple serogroups. Immunogenicity, antibody persistence, and safety of the EU-licensed meningococcal ACWY-tetanus toxoid conjugate vaccine (MenACWY-TT) were evaluated in healthy participants aged 11–55 years from the Philippines and Saudi Arabia.In this phase IIb, open, controlled study, 500 participants were randomised (3:1) to receive one dose of MenACWY-TT or a licensed meningococcal polysaccharide vaccine (Men-PS). Functional antibody responses against meningococcal serogroups A, C, W-135, and Y were assessed by a serum bactericidal antibody assay using rabbit complement (rSBA) at Month 0, Month 1, Year 1, Year 2, and Year 3. Vaccine response was defined as an rSBA titre ≥32 at Month 1 in participants who were seronegative (rSBA titre <8) pre-vaccination and as at least a four-fold increase in titre in participants who were seropositive pre-vaccination. Solicited symptoms were recorded up to Day 4, safety outcomes up to Month 6, and serious adverse events related to vaccination up to Year 3.Pre-specified criteria for non-inferiority of MenACWY-TT versus Men-PS were met in terms of rSBA vaccine response and incidence of grade 3 general symptoms. At Month 1, 82.7%–96.3% of MenACWY-TT and 69.7%–91.7% in Men-PS recipients had a vaccine response for each serogroup. At Year 3, ≥99.1% and ≥92.9% of MenACWY-TT recipients retained rSBA titres ≥8 and ≥128, respectively, as compared to ≥86.7% and ≥80.0% in the Men-PS group. Both vaccines had a clinically acceptable safety profile, although injection site redness and swelling were more frequent in MenACWY-TT recipients.These results suggest that MenACWY-TT could protect adolescents and adults against meningococcal disease up to three years post-vaccination.http://www.clinicaltrials.gov/NCT00356369.This study is registered at  Neisseria meningitidis is a major cause of endemic and epidemic invasive bacterial disease worldwide and is associated with high morbidity ; MenACWY-TT) has been recently approved by the European Medicines Agency for individuals aged 12 months and above. Previous studies have shown that MenACWY-TT is immunogenic and well-tolerated in toddlers, young children, adolescents, and young adults  at Month 0 to 10.01 [95% CI: 8.34 – 12.03] at Month 1). As expected, the percentage of participants with anti-TT concentrations ≥0.1 IU/mL (60.0% [95% CI: 50.2-69.2] at Month 0 and 56.6% [95% CI: 47.0–65.9] at Month 1) and the anti-TT GMCs (0.28 [95% CI: 0.20–0.39] at Month 0 and 0.27 [95% CI: 0.19–0.38] at Month 1) remained stable between pre- and post-vaccination in the Men-PS group.During the four-day post-vaccination period, grade 3 general symptoms were reported by five participants (1.3%) in the ACWY-TT group and by no participant in the Men-PS group Table . The difDuring the four-day follow-up after vaccination, pain was the most frequently reported solicited local symptom in both groups (in 143/370 [38.6%] participants in the ACWY-TT group and 40/124 [32.3%] participants in the Men-PS group). The most common solicited general symptom was headache in both groups, which was reported by 65/371 (17.5%) participants in the ACWY-TT group and 15/125 (12.0%) participants in the Men-PS group. Injection site redness and swelling were more frequently reported in the ACWY-TT group (15. 4% and 11.4% respectively) than in the Men-PS group (6.5% and 3.2% respectively)  and one participant reported two SAEs . The SAEs were reported in the ACWY-TT group, but none were considered related to vaccination by the investigators. No participants reported NOCIs or SAEs that were considered related to vaccination or events related to the lack of vaccine efficacy up to three years after vaccination. In the persistence phase of the study, two participants of the ACWY-TT group died due to a traffic accident (one subject between Year 1 and Year 2 and a second subject between Year 2 and Year 3).Non-inferiority of the immunogenicity of the MenACWY-TT vaccine as compared to the MenACWY polysaccharide vaccine in terms of rSBA vaccine response to the four meningococcal serogroups was demonstrated. At one month post-vaccination, rSBA titres ≥128, which is the conservative threshold for protection against meningococcal diseases, were reached for each of the four serogroups by at least 99.4% and 98.2% of the participants vaccinated with MenACWY-TT and the MenACWY polysaccharide vaccine, respectively. This observation is consistent with the results of previous studies in which the immune response induced by MenACWY-TT was evaluated in various age groups . Moreove197) induce higher antibody titres than plain polysaccharide vaccines in children, adolescents, and adults [Exploratory analyses showed that the rSBA GMTs for each meningococcal serogroup were statistically significantly higher at one month post-vaccination in participants vaccinated with MenACWY-TT compared with those who received the MenACWY polysaccharide vaccine, although no differences were detected in terms of percentage of participants with rSBA titres ≥8 or ≥128. These results are consistent with those of previous studies conducted on the MenACWY-TT vaccine in children 3–5 years of age  and in ad adults ,26,41-43N. meningitidis and acquisition of functional antibodies due to natural immunity may vary by location as meningococcal disease epidemiology varies substantially by geographic area [In this study, high rSBA titres were measured in both groups at pre-vaccination. This was expected for the age groups studied (adolescents and adults) because immunity to meningococcal strains is known to increase in an age-dependent manner . Moreovehic area . Vaccinehic area ,29,33,34Non-inferiority of the MenACWY-TT vaccine over the MenACWY polysaccharide vaccine, in terms of incidence of solicited and unsolicited grade 3 general symptoms reported within four days after vaccination, was demonstrated. Of note, clinically relevant severe general symptoms and vaccine-related events are most likely to occur during the four-day post-vaccination period. However, the incidence of any grade 3 symptoms seemed higher in the participants who received the MenACWY-TT vaccine than the MenACWY polysaccharide vaccine and this observation was driven by the local injection site reactions. The specific solicited injection site redness and swelling with any intensity were also more frequently reported in the participants who received the MenACWY-TT vaccine than the MenACWY polysaccharide vaccine. These observations are likely due to the TT content of the conjugate vaccine and are consistent with previous studies showing that local reactions are more frequent in individuals vaccinated with quadrivalent meningococcal conjugate vaccines compared to plain polysaccharide vaccines ,33,34,43The present study was limited by its open design due to the differences in the appearance and administration mode of the two study vaccines. The open design would not have influenced the immunogenicity results because the laboratory personnel was blinded during serological testing but had the potential to bias the safety reporting in favour of the control plain polysaccharide vaccine versus the meningococcal conjugate vaccine. The interpretation of the results has also been limited by the absence of validated correlates of protection for serogroups A, W-135, and Y and by the numerous exploratory statistical comparisons, which should be interpreted with caution. The generalizability of our results was limited by the small sample size and by the fact that the study was conducted in two countries. Other studies are being conducted in parallel to evaluate the immunogenicity and safety of the MenACWY-TT vaccine in younger age groups and to compare this vaccine with other licensed quadrivalent meningococcal conjugate vaccines . Of noteThis study showed that the MenACWY-TT vaccine, when administered as a single intramuscular dose to adolescents and adults 11–55 years of age, was non-inferior to a licensed meningococcal polysaccharide vaccine in terms of both immunogenicity and safety endpoints. Furthermore, MenACWY-TT induced an immune response against meningococcal serogroups A, C, W-135, and Y, which persisted up to three years after vaccination; rSBA GMTs for serogroups A, W-135, and Y were statistically significantly higher compared with those in participants who received the MenACWY polysaccharide vaccine, while comparable rSBA GMTs for serogroup C were measured in both vaccine groups. Both vaccines were well-tolerated and their safety and reactogenicity profiles were comparable, except for higher reported rates of injection site redness and swelling after vaccination with MenACWY-TT.Mencevax and Nimenrix are trademark of the GlaxoSmithKline Group of companies.ATP: According to protocol; CI: Confidence interval; ELISA: Enzyme-linked immunosorbent assay; GMT: Geometric mean antibody titres; GMC: Geometric mean antibody concentration; LL: Lower limit; UL: Upper limit; MenACWY-TT: Meningococcal quadrivalent serogroups A, C,W-135 and Y vaccine with all serogroups conjugated to the tetanus toxoid (TT) carrier protein; Men-PS: Meningococcal quadrivalent polysaccharide vaccine; NOCI: New onset of chronic illness; rSBA: Serum bactericidal antibody assays using baby rabbit serum as exogenous complement source; (S)AE: (Serious) adverse event; TT: Tetanus toxoid.Research Institute for Tropical Medicine, Philippines) and Dr Ziad A. Memish  received grants from the GlaxoSmithKline group of companies. Dr Charissa Borja-Tabora received support for meetings, travel or accommodation expenses from the GlaxoSmithKline group of companies in the past 5 years. Veronique Bianco and Drs Marie Van der Wielen, Dominique Boutriau and Jacqueline Miller are employees of GlaxoSmithKline group of companies. Drs Marie Van der Wielen and Dominique Boutriau declare stock ownership in the GlaxoSmithKline group of companies. Dr Jacqueline Miller declares restricted share in the GlaxoSmithKline group of companies. Dr Dominique Boutriau is also inventor of certain patents of the GlaxoSmithKline group of companies. Dr Cecilia Montalban declares that she has no competing interests.The institutes of Dr Charissa Borja-Tabora  and VB (biostatistician) were employed by GlaxoSmithKline group of companies and involved in all stages of the study . All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/116/prepubPercentage of participants per age strata with an rSBA vaccine response.Click here for filePercentage of participants per age strata with rSBA titres ≥1:8 and 1:128 and GMTs.Click here for file


Inflammation and oxidative stress are associated with human health and the disease status. The present study aimed to investigate the longitudinal correlation between the diacron reactive oxygen metabolites (d-ROMs) level, as an oxidative stress-related marker, and high-sensitivity C-reactive protein (hsCRP), as an inflammatory marker, during a one-year period among asymptomatic subjects.The data, including anthropometric and biochemical markers, were collected at baseline and after the one-year period from 71 participants . The correlation between the changes of the d-ROMs and hsCRP levels during the study period was examined.A simple correlation analysis showed a significant and positive correlation to exist between the changes of the d-ROMs and hsCRP levels . This significant correlation remained independent in a multiple linear regression analysis adjusted for confounding factors.The present findings suggest that the relationship between the d-ROMs and hsCRP levels could be prospectively followed, and that monitoring both markers may help to better understand the cooperation of inflammation and oxidative stress in association with health and disease. Further studies are necessary to clarify the biological mechanism(s) responsible for the observed relationship.Oxidative stress; Oxygen reactive species; Inflammation; CRP In recent years, chronic inflammation, as assessed by the C-reactive protein (CRP) level, and oxidative stress, as assessed by oxidative stress-related markers, have attracted much attention as non-traditional pathological factors, since these conditions have been shown to play important roles in human health and disease . While tThe diacron reactive oxygen metabolites (d-ROMs) test was recently introduced, and this measurement can quantify the oxidative stress status by measuring the levels of hydroperoxides of global organic compounds  ,8. This This one-year observational design study consisted of 71 asymptomatic subjects  who had health check-up examinations both at baseline and after the one-year period. The inclusion criteria were non-smokers and participants not taking any medications or supplements. The exclusion criteria were participants who had acute infectious diseases, such as a common cold, or who had a history of cardio/cerebrovascular, thyroid, collagen, severe kidney or liver diseases. This study was approved by the Institutional Ethics Committee, and all subjects gave their informed consent.2O2.At baseline and after the one-year period, we obtained data from each subject in an overnight fasted state. The body mass index (BMI) was calculated based on the weight and height measured while subjects wore light clothing without shoes. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured in the subject’s right arm with a mercury sphygmomanometer while the subject was the seated position, and then, the mean blood pressure (MBP) was calculated by the equation of DBP + [(SBP-DBP)/3]. The serum total cholesterol (TC), triglyceride (TG), HDL-C and fasting plasma glucose (FPG) levels were measured by standard enzymatic methods. The serum hsCRP levels were measured by an enzyme-linked immunosorbent assay . The d-ROMs levels were measured using a kinetic spectrophotometric assay  with intra- and inter-assay coefficients of variation of 2.1% and 3.1%, respectively ,8. BriefThe data are expressed as the means ± standard deviation (SD) or the medians plus the interquartile range. The data between at baseline and after the one-year period were compared using paired t-tests. Changes in the respective measurements during the one-year period were calculated by subtracting the values at baseline from the values after the one-year period. A simple correlation test (Pearson’s test) and a multiple linear regression analysis, adjusted for all the other factors, were used to observe the correlation between the changes of the d-ROMs and hsCRP levels during the one-year period. The TG and hsCRP values were log-transformed in these analyses because of their skewed distributions. Statistical significance was defined as a P-value < 0.05.2 in the BMI (P = 0.29), 93 ± 13 to 93 ± 11 mmHg in MBP (P = 0.52), 5.32 ± 1.33 to 5.25 ± 0.91 mmol/L in TC (P = 0.51), 1.61 ± 0.41 to 1.62 ± 0.43 mmol/L in HDL-C (P = 0.42), 0.92 (0.71-1.39) to 1.02 (0.67-1.49) mmol/L in TG (P = 0.59), 5.38 ± 0.54 to 5.52 ± 0.77 mmol/L in FPG (P = 0.06).After the one-year period, the hsCRP levels showed a small but significant increase , while the d-ROMs levels showed a non-significant and slight increase . The mean/median levels of the other measured factors between the values at baseline and post-one-year period were largely unchanged: with changes from 22.5 ± 3.0 to 22.6 ± 3.1 kg/mA simple correlation analysis showed that there was a significant and positive correlation between the changes of the d-ROMs and hsCRP levels . The chaThe present study found that there was an independent, significant and positive correlation between the changes of the d-ROMs and hsCRP levels during a one-year period among asymptomatic subjects. Although the cooperation of oxidative stress and inflammation is a recent key concept developed as a result of a deeper understanding of the pathophysiology of health and disease , the corAlthough the detailed biological mechanism (s) underlying the present results remain unclear, there are several possible explanations. Oxidative stress, which results from an imbalance in the oxidative/antioxidative system , and inflammation can both induce an oxidative stress response and inflammatory molecules via cell dysfunction in systemic organs, including the adipose tissue, liver and vasculature ,3. FurthThere were some limitations to the present study. The observational period was relatively short, while this can have merit because it can reduce the impact from the fluctuation of various factors influencing inflammation and oxidative stress . The sample size was small, and the study population was basically restricted to the non-severe diseased participants. Future studies with a long-term prospective design and with larger and more varied populations are needed.In summary, the present study confirmed that there was an independent, significant and positive correlation between the changes of the d-ROMs and hsCRP levels during a one-year period among asymptomatic subjects. The relationship between the d-ROMs and hsCRP levels was prospectively followed, and monitoring both markers may help to better understand the cooperation of inflammation and oxidative stress in association with health and disease. Further studies are necessary to clarify the biological mechanism (s) underlying the observed relationship.


One hour after the radiolabeled peptide or FDG was given, micro-PET/computed tomography images were acquired. Thereafter, the uptake of the 68Ga or 18F in dissected tissue was determined.Two groups of female BALB/c nude mice were inoculated with LS174T human colonic tumor cells i.p. One group received 5 MBq 68Ga-labeled peptide in the tumor lesions (23.4 ± 7.2% ID/g) and low background activity levels were observed . This resulted in a clear visualization of all intra-abdominal tumor lesions ≥ 10 μL and even some tumors as small as 5 μL (2 mm diameter). 18F-FDG efficiently localized in the tumors (8.7 ± 3.1% ID/g) but also showed physiological uptake in various normal tissues .Within 1 h, high uptake of the 68Ga-labeled peptide could be a very sensitive imaging method for imaging colonic cancer, disclosing occult lesions.Pretargeted immuno-PET with bsmAb and a  Colorectal cancer is a frequently diagnosed cancer type. It is the third most common cancer in both men and women in the Western world ,2. The oSpecific detection of malignant colorectal tumor lesions could be achieved by (pretargeted) antibody-guided radionuclide imaging. The combination of the specificity of antibody targeting and the sensitivity of positron-emission tomography [PET] is very promising. Radiolabeled antibodies have been tested for the detection of several cancer types. However, imaging with radiolabeled whole antibodies requires a relatively long interval between injection and imaging acquisition for adequate contrast to develop due to the slow accretion of intact antibodies in tumors and their slow clearance . PretargCoupling two haptens together improves peptide uptake and stability by a process known as affinity enhancement . ChelateFludeoxyglucose [FDG]-PET/computed tomography [CT] has an established role in the work-up of patients with metastasized colorectal cancer and could change patient management in > 25% of patients -16. Othe68Ga-labeled peptide, IMP288. Pretargeted immuno-PET was compared to 18F-FDG-PET in a preclinical orthotopic model in mice with small, intraperitoneally growing CEA-expressing colonic tumor lesions.In the present study, we examined the sensitivity of pretargeting with a bispecific monoclonal anti-carcinoembryonic antigen [CEA] × antihistamine-succinyl-glycine [HSG] antibody, TF2, and a 68Ga-IMP288 peptide. IMP288 was synthesized and purified as described by McBride et al. -D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2.The bsmAb, TF2, and the peptide IMP288 were provided by Immunomedics . The preparation of TF2 and binding properties has previously been described -29. Gel e et al. . IMP288 125I  by the iodogen method as described previously [125I-labeled TF2 was purified by eluting the reaction mixture with phosphate-buffered saline [PBS] and 0.5% w/v bovine serum albumin [BSA]  on a PD-10 column . IMP288 was labeled with 68Ga as described previously . The mice were fasted for 10 h before the 18F-FDG injection, anesthetized, and kept warm at 37°C. The mice were euthanized 1 h after the injection of 68Ga-IMP288 or 18F-FDG by CO2/O2 asphyxiation, followed by cardiac puncture to obtain blood.Female nude BALB/c mice (6 to 8 weeks old), weighing 20 to 25 g, received an intraperitoneal injection of 0.5 mL of a suspension of 1 × 10eviously . The oth68Ga-IMP288 or 18F-FDG with an Inveon animal PET/CT scanner  having an intrinsic spatial resolution of 1.5 mm  was calculated. The correlation between the weight and uptake of Immunohistochemical analysis of CEA was performed on 4-μm-thick formalin-fixed, paraffin-embedded tissue sections. The sections were deparaffinized in xylol and rehydrated through a graded ethanol into water series. To block endogenous peroxidase, slides were blocked with 3% hydrogen peroxide in phosphate buffered saline (10 min at room temperature). Then sections were blocked with 20% normal goat serum  in 1% BSA-PBS (30 min at room temperature [RT]). Subsequently, tumor sections were incubated with a 1:12,000 dilution of polyclonal rabbit anti-CEA antibody  overnight at 4°C, followed by incubation with a goat-anti-rabbit biotinylated secondary antibody (1/200 in 1% BSA-PBS)  for 30 min at RT. Finally, avidin-biotin-enzyme complex (Vector Laboratories Inc.) was applied for 30 min at 37°C, and 3,39-diaminobenzidine was used to develop the tumor sections. Human colon carcinoma was used as a positive control, and substitution of the primary antibody with 1% BSA-PBS was used as the negative control.PET/CT images were scored by a blinded, independent, experienced nuclear physician (W.O.), being asked to record the presence of intra-abdominal tumor lesions. When lesions were present, he was asked to draw a region of interest [ROI] around the tumor. Each lesion was given a number on a 1 to 3 scale that defined the reader's confidence that the uptake was related to a tumor . The imaging findings were then compared with the tumor lesions found at dissection. The detection rates for tumors < 10 μL and ≥ 10 μL were calculated, corresponding with a sphere diameter of < 2.7 or ≥ 2.7 mm, respectively.p < 0.05.Statistical analysis was performed using the SPSS software  and GraphPad Prism version 5.00 for Windows . Means and standard deviations were used to describe continuous data, unless stated otherwise. Correlations were determined using a Spearman's correlation test. The level of significance was set at n = 10/mouse; range, 4 to 17). Most frequent localizations were at the rectovesical pouch, the mesentery, and the subhepatic, -splenic, and -phrenic spaces. Some tumor nodules were adjoining in groups of two or three lesions. Three-dimensional caliper measurements indicated that the maximum diameter of the tumor lesions varied between 1 and 15 mm , and weights varied between 0.3 and 650 mg .Three weeks after the intraperitoneal injection of the LS174T cells, the mice did not show clinical signs of discomfort or change in body weight. At dissection, the abdomen contained multiple solid tumor lesions .The biodistribution of 18F-FDG localized efficiently in the tumors .68Ga-IMP288 resulted in a clear delineation of the tumors. An example of a PET/CT image is shown in Figure Immuno-PET with TF2 and Interestingly, one lesion that was macroscopically doubtful to be a tumor, and showing minimal uptake on immuno-PET, had an activity concentration as low as 0.49% ID/g. This uptake level was much lower than that of the other lesions in the same animal . This lesion with the low uptake was shown by immunohistochemistry to consist > 90% of necrotic tissue and infiltrated leukocytes, lack CEA expression, and have only a small rim of vital tumor cells  might have limited image resolution. Visser et al. [68Ga. Combined with the partial volume effect, this could explain the lower detection rate of the smallest tumor lesions with pretargeted immuno-PET despite the higher radioactivity concentration of TF2 and 68Ga-IMP288 in the smaller tumors.For clinical application, r et al.  showed t18F, the most widely used positron-emitting radioisotope, would be suitable due to its short positron range in the tissue (0.62 mm), which might increase the image resolution. McBride and, subsequently, Laverman et al. developed an innovative and rapid method for labeling peptides with 18F based on a metal chelator [Due to the flexibility of the di-HSG peptides, the use of other PET radionuclides for this pretargeting system can be explored. chelator ,47. The chelator . Transla68Ga-IMP288 is a specific and sensitive method for detecting colon cancer in a preclinical model. Further clinical trials should focus on the diagnostic accuracy of pretargeted immuno-PET and determine its additional value in the clinical setting.In summary, this study indicates that pretargeted immuno-PET with TF2 and WJM, DMG, and C-HC are employed by or have financial interest in Immunomedics, Inc. and/or IBC Pharmacauticals, Inc. The other authors declare that they have no competing interests.RS conceived the study, carried out the imaging studies and the analysis of the studies, and drafted the manuscript. WTAG conceived the study and helped draft the manuscript. RMS participated in the design of the study and helped draft the manuscript. GMF carried out the labeling procedures and the imaging studies. WJM and C-HC synthesized and purified the pretargeting agents. PL carried out the imaging studies and helped with the analysis of the studies. DMG participated in the design of the study and helped draft the manuscript. WJGO carried out the analysis of the imaging studies and helped draft the manuscript. OCB conceived the study, participated in the design of the study, and helped draft the manuscript. All authors read and approved the final manuscript.


S. pyogenes use this cyclic nucleotide derivative as a second messenger instead of c-di-GMP, a well-studied important bacterial second messenger. GdpP, c-di-AMP phosphodiesterase, is responsible for degrading c-di-AMP inside cells, and the cellular role of GdpP in S. pyogenes has not been examined yet. To test the cellular role of GdpP, we created a strain with a nonpolar inframe deletion of the gdpP gene, and examined the properties of the strain including virulence. From this study, we demonstrated that GdpP influences the biogenesis of SpeB, the major secreted cysteine protease, at a post-translational level, susceptibility to the beta lactam antibiotic ampicillin, and is necessary for full virulence in a murine subcutaneous infection model.Small cyclic nucleotide derivatives are employed as second messengers by both prokaryotes and eukaryotes to regulate diverse cellular processes responding to various signals. In bacteria, c-di-AMP has been discovered most recently, and some Gram-positive pathogens including  Pseudomonas aeruginosa, c-di-GMP has an important role in regulating virulence factor expression and biofilm formation [Staphylococcus aureus, Streptococcus pyogenes, and Listeria monocytogenes synthesize c-di-GMP. Furthermore, Staphylococcus and Streptococcus do not even possess enzymes with domains responsible for c-di-GMP synthesis  or hydrolysis (an EAL or HD-GYP phosphodiesterase domain) [In bacteria, cyclic dimeric guanosine 3’,5’-monophosphate (c-di-GMP) has recently emerged as an important second messenger regulating gene expression . In humaormation . However domain) . Recent  domain) .Staphaureus [Mycoplasma genitalium [Mycobacteriumpulmonis [Streptococcspneumoniae [L. monocytogenes [S. pyogenes, also has one DAC protein (Spy_1036). The c-di-AMP synthesized by the DacA protein is degraded by a membrane-anchored intracellular protein GdpP [in vivo and in vitro [gdpP gene increases the concentration of c-di-AMP more than 10 fold in Staphaureus [B. subtilis [Staphaureus, gdpP deletion not only suppresses the growth defect caused by stress on envelope associated with an inability to synthesize lipoteichoic acid, but also increases the degree of peptidoglycan cross-linking, decreases susceptibility to cell wall targeting antibiotics, and reduces cell size by ~ 20% [B. subtilis, the deletion of the gdpP ortholog, yybT, increases sporulation efficiency of cells challenged with the DNA gyrase inhibitor nalidixic acid, and decreases the susceptibility to beta-lactam antibiotics [S. pyogenes has not yet been elucidated.c-di-AMP is synthesized by a protein DacA, which contains a di-adenylate cyclase domain (DAC domain) –8. The paureus , Mycoplanitalium , Mycobulmonis , Strepumoniae , and L. ytogenes  that posein GdpP . GdpP haein GdpP ,5 . SpeB is a cysteine protease encoded on a chromosomal gene [mal gene , so mostmal gene . Potentimal gene , or indimal gene . SpeB hamal gene , and kinmal gene . SpeB remal gene . Liberatmal gene . Throughcis-trans isomerases [SpeB production is regulated transcriptionally and post transcriptionally. Initially, SpeB is translated as a preproprotein (43 kDa) containing a signal sequence and a pro region of SpeB. When SpeB is secreted by the Sec system, the signal sequence is cleaved off to form the proSpeB zymogen (~40 kDa), which is still an inactive protease. After being secreted, the zymogen is folded and processed to the active protease (23 kDa) by multiple cleavages in the pro region. Previous studies showed that this processing is influenced by peptidyl-prolyl omerases –24, and omerases ,26.S. pyogenes, we deleted the gdpP gene (spy_2184) and examined the deletion effect. Here, we provide the evidence that the c-di-AMP phosphodiesterase GdpP is required for the processing of SpeB and full virulence of S. pyogenes in an animal infection model.In this study, to investigate the role of GdpP in the physiology and virulence of S. pyogenes HSC5 [S. pyogenes in liquid, two media, THY and Cmedium, were used. THY medium, Todd-Hewitt medium (BBL) supplemented with 0.2% yeast extract (Difco), was used for routine culture, and Cmedium was exclusively employed to determine SpeB expression [2 and 15% CO2) employing a commercial gas generator . For plasmid construction, Escherichia coli DH5α [E. coli; kanamycin 50 µg/ml for E. coli and 500 µg/ml for S. pyogenes; erythromycin 500 µg/ml for E. coli and 1 µg/ml for S. pyogenes; chloramphenicol 7 µg/ml for E. coli and 3 µg/ml for S. pyogenes.nes HSC5  was usedpression . Liquid oli DH5α  or TOP10S. pyogenes by electroporation as described previously [™ Bacterial Genomic DNA Kit (Sigma) was used to purify chromosomal DNA of S. pyogenes. When required, DNA fragments were purified using Mini Elute™ gel extraction kit (Qiagen) following agarose gel electrophoresis.Plasmid DNA was isolated by standard techniques and used to transform eviously . RestricS. pyogenes cultures was isolated through the Qiagen RNeasy mini kit (Qiagen) combined with mechanical disruption of cells [® Tag (Invitrogen) was used to synthesize cDNA from RNA and to amplify the cDNA by PCR. In order to detect the transcript containing the intergenic region of gidA and gdpP, gdpP and rpl9, or rpl9 and holB, the primer sets of gidA-gdpP-f (gtaagccagaacttattttgaaacg) and gidA-gdpP-r (gatcaaattgaactactccaacagg), gdpP-rpl9-f (atcacagaagagatgatgattttcc) and gdpP-rpl9-r (atttcacaggaacttcaattagacc), or rpl9-holB-f (ctcaagctgaaatattagcagaagc) and rpl9-holB-r (agcaattgttacattattccaatcc) were used. These primer sets were supposed to produce about an 1 kb PCR product if an appropriate template exists.RNA from of cells . RNA quagdpP was created by the method previously described [ggatccccaacgctgtagaatggtataatcc) and GdpPInframeEx-r (cgggatccctaatacaaagctagcctcaacgtg) were used to amplify an 1.55 kb internal fragment of gdpP. (The BamHI restriction sequence in the primers is underlined.) This amplified fragment was inserted into the BamHI restriction site in the pCRII vector (Invitrogen). The resulting plasmid was then used as a template in an ‘inside-out’ PCR reaction with the primers of GdpPInframeIn-f (ccatcgattagttgatcatcacagaagagatgatg) and GdpPInframeIn-r (ccatcgatcaccaaaagaaattcctatgctgag). Cleavage of this fragment with ClaI followed by subsequent re-ligation resulted in a nonpolar inframe deletion that replaces DNA sequence encoding E270 – V440 of GdpP with the sequence of gatcgatta encoding DRL. The inframe deletion allele of gdpP was then inserted into the BamHI restriction site in the S. pyogenes-E. coli shuttle vector, pJRS233. The generated plasmid, pJRS233::gdpP-IFD, was used to replace the wild type gdpP with gdpP∆270-440 by a method that employs the temperature sensitivity of the plasmid replication origin as was described previously [gdpP-IFD, was introduced into the wild type HSC5 by electroporation, and transformants were grown in the THY medium containing erythromycin at 30°C. Then, the plasmid was inserted into the chromosome through a homologous recombination by shifting temperature to 39°C. One colony growing at 39°C in the presence of erythromycin was picked and grown at 30°C in the THY medium without erythromycin. This temperature shift selects for the excision of the plasmid through another homologous recombination, which could leave either the wild type or deletion allele of the gdpP gene on the chromosome depending on the location of the crossover. Since both the wild type revertant and the inframe deletion mutant went through the same process, we used the wild type revertant as the wild type control strain for virulence study using a mouse subcutaneous infection model. The allelic replacement was confirmed by PCR and sequence analysis.An inframe deletion allele of escribed . Brieflyeviously . BrieflygdpP gene was inserted into an expression vector pABG5, an E. coli - S. pyogenes shuttle vector that has the promoter of rofA (regulator of fibronectin binding protein A), which has been successfully used to express streptococcal genes previously [gdpP gene was PCR-amplified with primers of GdpPcomp-f (aaaaaagcatgcgagtcaatcctgcggacatc) and GdpPcomp-r (aaaaaaggcaccagcatagcctgttgggacc) and digested with the restriction enzymes, SphI and BanI (Their restriction sequences in the primers are underlined.). The amplified PCR product (~2.2 kb) was then inserted downstream of the rofA promoter in pABG5. To create GdpP∆TM, GdpP with the deletion of the transmembrane domain, the GdpP expression plasmid was PCR-amplified with primers of GdpP∆TM-f (gctctagacatctttataacctcttaagcacac) and GdpP∆TM-r (gctctagacaaaaagaagcttatcaattatc), then the inverse PCR product was digested with XbaI and re-ligated (The restriction sequence in the primers is underlined.). The expression of GdpP and GdpP∆TM was confirmed by Western blotting. The expression level of these proteins in various strains was compared to GdpP expression in the wild type by measuring band intensities in digital images of Western blots analyzed with NIH ImageJ software (http://rsbweb.nih.gov/ij/).To express GdpP ectopically, the eviously ,33. FirsCmedium was used to derive background values. To ensure that all proteolytic activity was specifically the result of SpeB, the cysteine protease-specific inhibitor E-64  was added to selected samples. This treatment typically reduced activity by >95%. The presence of the pro-protein and processed forms of SpeB was determined through Western blot analysis as described previously [600. After taken at each time point, the cultures were adjusted to 0.5 of OD600 with fresh medium and cells were removed by filtration (0.45 µm of pore size). Then, 10 µl of each culture supernatant was applied to each lane of a SDS-PAGE gel. The amount of the culture supernatants for the proteolytic activity assay was normalized in the same way.The proteolytic activity of culture supernatants was quantitated by the method of Hauser et al. , which meviously . The loaS. pyogenes strains to cause disease in soft tissue was evaluated by subcutaneous infection of 6-8 week old SKH1 hairless mice (Charles River Labs) as described previously [S. pyogenes strains were grown in 10 ml THY media to a mid-logarithmic phase (0.30 of OD600), washed, and resuspended in 1ml of sterile saline. This suspension was subjected to brief sonication (3 or 4 pulses of 10 sec each) to disrupt streptococcal chains, and even disruption was confirmed through microscopy. The bacteria were diluted in saline to obtain the appropriate concentration of viable streptococcal cells, which was confirmed by determination of the number of colony forming units (CFU). Then, each mouse received a subcutaneous injection of 107 CFU in a 100 µl volume into the right flank. Each S. pyogenes strain was analyzed in a group of 10 mice. The area of the draining ulcers that formed was documented every 24 hrs by digital photography and the precise area contained by each ulcer was calculated from the digital record. Any differences in the areas of ulcers between experimental groups were tested for significance by the Mann–Whitney U-test [P values were <0.05. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of Southern Illinois University (SIU). All mice were anesthetized with isoflurane when the lesion sizes were measured and euthanized by CO2 asphyxiation at the end of the experiment.The ability of eviously . Brieflyy U-test . The nulgdpP gene is located in the middle of genes with the same orientation  and holB encoding DNA polymerase III delta prime subunit follow gdpP. To investigate whether gdpP is expressed as a member of a multigene operon or independently, reverse transcriptase-PCR (RT-PCR) was performed  and 0.55 hr-1 ± 0.01 in THY medium and 0.64 hr-1 ± 0.01 and 0.71 ± 0.01 in Cmedium, respectively. In both media, the significance values  determined by two-tailed paired Student’s t test between the growth rates of ∆GdpP and the parental strain were greater than 0.05, indicating that their growths were not significantly different.To delete the Cmedium culture supernatant of the gdpP deletion strain, ∆GdpP, to that of the wild type strain HSC5. The culture supernatant of ∆GdpP showed much less protease activity of SpeB than that of the wild type  to cause disease in soft tissue was evaluated using a murine subcutaneous infection model. In this assay, the ulcer size caused by ∆GdpP was compared to that caused by the wild type control strain that is a wild type revertant strain obtained from the inframe deletion procedure. At the last step of inframe deletion, some strains acquire the inframe deletion allele and the others retain the wild type genotype depending on the location of homologous recombination. During the several passages of cultures in the inframe deletion process, we experienced that S. pyogenes could loose M protein, a cell surface-anchored major virulence factor that influences virulence in this animal model. Thus, we confirmed M protein expression from both the wild type revertant control and ∆GdpP using Western blotting (data not shown). When we compared the ulcer sizes at 3 days post-infection when ulcer formation was maximal in both strains, ∆GdpP caused smaller lesion sizes than the wild type revertant . We do not know the biological significance of the coexpression of these genes, but the activity of GdpP may influence the fidelity of replication through HolB or of translation through Rpl9.Our RT-PCR revealed that the troscopy . The ribibosomes , the locribosome . The nex E. coli , a S. pygdpP gene in Staphaureus and B. subtilis increases the degree of peptidoglycan cross-linking and resistance to beta-lactam antibiotics [gdpP gene in S. pyogenes also increased the resistance to the beta-lactam antibiotic, ampicillin, suggesting that GdpP in S. pyogenes could also influence the cross-linking status of the cell wall. Most secreted proteins in S. pyogenes are exported through the general secretory (sec) pathway. SpeB is secreted through Exportal, which is a general sec system forming in a distinct cellular membrane microdomain [gdpP disruption might disturb the formation of Exportal or mislocate HtrA, which could influence SpeB processing in turn. Another possibility is that the increased amount of c-di-AMP due to the absence of GdpP might influence the biogenesis of RopA or PrsA. Both proteins are peptidyl-prolyl cis-trans isomerases (PPIase) required for the full conversion of proSpeB to active SpeB [There are several ways that a SpeB zymogen is converted to active SpeB. A zymogen can cleave the pro region by itself in a reduced condition ,42. Howeibiotics . The delrodomain  containirodomain . HtrA, arodomain ,26, so tive SpeB –24. The ive SpeB .gdpP in the chromosomal gdpP deletion mutant restored SpeB activity to more than the wild type level (GdpP is a membrane-anchored protein. It has a non-cleavable signal sequence containing two membrane-spanning helixes at the N-terminus. Thus, GdpP hydrolyzes c-di-AMP near the cell membrane. The multi-copy expression of gdpP gene attenuates the virulence of S. pyogenes in a murine model of subcutaneous infection. Probably, the processing defect of SpeB in the ∆GdpP contributes to the attenuation since removing protease activity from SpeB in HSC5 attenuates virulence in the same infection model. The strain JWR10 is a HSC5 mutant in which an amino acid residue in the active site of SpeB, cysteine 192, is replaced with serine [The deletion of the h serine . This mugdpP gene in the human pathogen S. pyogenes impairs the processing of SpeB, decreases sensitivity to the beta-lactam antibiotic ampicillin, and attenuates virulence in a murine model of subcutaneous infection, indicating that GdpP has an important role in maintaining S. pyogenes virulence.In this study, we demonstrated that the deletion of the


Arcobacter nitrofigilis  Vandamme et al. 1991 is the type species of the genus Arcobacter in the family Campylobacteraceae within the  Epsilonproteobacteria. The species was first described in 1983 as Campylobacter nitrofigilis [1] after its detection as a free-living, nitrogen-fixing Campylobacter species associated with Spartina alterniflora Loisel roots [2]. It is of phylogenetic interest because of its lifestyle as a symbiotic organism in a marine environment in contrast to many other Arcobacter species which are associated with warm-blooded animals and tend to be pathogenic. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a type stain of the genus Arcobacter. The 3,192,235 bp genome with its 3,154 protein-coding and 70 RNA genes is part of the  Genomic  Encyclopedia of  Bacteria and  Archaea  project. T (= DSM 7299 = ATCC 33309 = CCUG 15893) is the type strain of the species Arcobacter nitrofigilis, which is the type species of the genus Arcobacter [T was isolated from roots of Spartina alterniflora Loisel (cordgrass) growing in salty marshes at the East coast of Canada. It was the first description of an organism in this kind of habitat that belonged to the genus Campylobacter, as described based on phenotypic and biochemical traits [nitrofigilis means 'nitrogen-fixing' and is based on the outstanding characteristic of this species [Arcobacter, meaning 'bow-shaped rod', was introduced in 1991 and its separation from the genus Campylobacter was based on DNA-DNA and DNA-rRNA hybridization [Arcobacter comprises nine species, some of which are associated with warm-blooded animals whereas others are found in marine environments.Strain CICampylobacteraceae several whole-genome sequences have already been deciphered: A. butzleri strain RM4018 [Arcobacter, as well as genomes from seven species of the genus Campylobacter, and Sulfurospirillum deleyianum [Within the leyianum .A. nitrofigilis are known in the literature, with F2176 and F2173 [Arcobacter share 93.8-94.6% 16S rRNA sequence identity with strain CIT, whereas the type strains from other genera in the family Campylobacteraceae share less than 89% sequence identity with strain CIT [A. nitrofigilis strain CIT, together with the description of the complete genome sequencing and annotation.Only few additional strains belonging to the species A. nitrofigilis strain CIT in a 16S rRNA based tree. The four 16S rRNA gene sequences in the genome differ from each other by up to two nucleotides, and differ by up to three nucleotides from the previously published 16S rRNA sequence (L14627) generated from CCUG 15893, which contains nine ambiguous base calls.A. nitrofigilis cells are Gram-negative, bow-shaped or curved rods of 1–3 µm length and 0.2–0.9 µm width , cis-9-hexadecenoic (cis-C16:1ϖ7c) and cis-9-octadecenoic acid (cis-C18:1ϖ7c) [The major respiratory quinones are menaquinone 6 and a second atypical menaquinone 6 that has not yet been clearly identified . The maj18:1ϖ7c)  Genomic  Encyclopedia of  Bacteria and  Archaea  project [This organism was selected for sequencing on the basis of its phylogenetic position , and is  project . The gen project  and the A. nitrofigilis strain CIT, DSM 7299, was grown on DSMZ medium 429 (Columbia agar including 5% horse blood) [et al. [e blood)  at 28°C. [et al. .http://www.jgi.doe.gov/. Draft assembly was based on 138 Mb 454 standard and 454 paired end data . Newbler  parameters are -consed -a 50 -l 350 -g -m -ml 20. The initial Newbler assembly contained 42 contigs in 3 scaffolds. It was converted into a phrap assembly by making fake reads from the consensus and collecting the read pairs in the 454 paired end library. Illumina sequencing data was assembled with Velvet [www.phrap.com) was used for sequence assembly and quality assessment in the following finishing process. After the shotgun stage, reads were assembled with parallel phrap . Possible mis-assemblies were corrected with gapResolution (http://www.jgi.doe.gov/), Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing [The genome was sequenced using a combination of Illumina and 454 technologies. An Illumina GAii shotgun library with reads of 50 Mb, a 454 Titanium draft library with average read length of 243 bases, and a paired end 454 library with average insert size of 24 kb were generated for this genome. All general aspects of library construction and sequencing can be found at h Velvet , and the bombing . Gaps beGenes were identified using Prodigal  as part The genome is 3,192,235 bp long and comprises one main circular chromosome with an overall G+C content of 28.4% . Of the


This study aims to validate the Chinese version of the Reynolds’ Suicidal Ideation Questionnaire (SIQ) in a Chinese society and explore a convenient short version.A sample of 711 cases was derived from two territory-wide surveys of Hong Kong adolescents aged between 15 and 19 years old.The SIQ and the Suicidal Ideation Questionnaire-Junior (SIQ-JR) demonstrated good reliability and concurrent validity among Hong Kong adolescents. However, the factor structure for both SIQ and SIQ-JR appeared to be unclear. A four-item short form of the SIQ-JR, namely, SIQ-JR-4, was proposed.The SIQ-JR-4 is an ideal substitute of the SIQ/SIQ-JR for future quick assessment of suicidal ideation in Chinese young adolescents. Suicide risk begins to increase since adolescent ages ,2. EarlyThe Reynolds’ Suicidal Ideation Questionnaire (SIQ) is a 30-item self-administered measurement for assessing severity of suicidal ideation among adolescents . The SuiIn this study, the SIQ/SIQ-JR has been translated into Chinese in accordance with social and culture context. It aims to shed light on the psychometric characteristics of the Chinese version of the SIQ and SIQ-JR and also explore a short form for fast screening purpose. A community-based sample of adolescents in Hong Kong was employed in the analysis.Data for the analysis of this study derived from two territory-wide surveys of Hong Kong residents. The sampling frame for both surveys was the Frame of Quarters, which is maintained by the Census and Statistics Department of Hong Kong. This is the most complete and up to date registry of residential addresses in the territory. Design of interviews and measurements used in both surveys were the same. Face-to-face interviews were carried out by trained interviewers. Sensitive questions like suicidal behaviors were self-reported by participants on a septe sheet of questionnaire.Slightly different sampling procedures were employed for generating two samples. Sample 1 was an extracted group of 203 respondents aged 15-19 years from a survey of local residents aged 15-59 years conducted between December 2003 and July 2004. First, a household was randomly selected from the Frame of Quarters, and second, one household member was randomly selected to participate in the study. This survey yielded successful interviews with 2,219 respondents, giving a response rate of 62%. Gender and age distributions in this yielding sample were similar to that in the general population. Sample 2 was generated from another survey of local residents aged 15-19 years conducted in June-August 2004. A septe list of households from the Frame of Quarters was prepared and all adolescents aged between 15 and 19 years in these households were invited to be interviewed. A total of 511 adolescents participated in the study, yielding a response rate of 61% by household. Both samples in total yielded 714 respondents, among which, 3 had missing values in the SIQ. Hence, there were 711 valid cases for the analysis of this study. Written informed consent was obtained from all respondents for both surveys. Details of the two household surveys have been published elsewhere . This stThe surveys began with a face-to-face interview, in which all participants answered interviewer-administered questions about depression, hopelessness, and anxiety. They were then asked to finish a self-reported questionnaire that included questions on suicidal ideation and suicide attempt.The self-reported booklet of questionnaire included a question “During the past 12 months, had you ever considered suicide?” and a question “During the past 12 months, had you ever attempted to commit suicide?” Participants with positive responses to these two questions were considered to have suicidal ideation and suicide attempt in the past 12 months, respectively.Severity of suicidal ideation was assessed by the Chinese version of Suicidal Ideation Questionnaire (SIQ), indicated by the frequency of suicidal ideation within the past month . The SIQThe severity of depressive symptoms was measured by the Chinese version of the Center for Epidemiologic Studies Depression (CES-D) Scale, a 20-item questionnaire ,18. RespHopelessness was assessed using the 20-item Chinese version of the Beck’s Hopelessness Scale (BHS) ,22. UnliThe severity of anxiety symptoms was measured by a 7-item anxiety subscale of the Chinese Depression Anxiety Stress Scales (DASS) ,25. It iThe Statistical Package for the Social Sciences (SPSS) for Windows, version 17.0 , Analysis of Moment Structures (AMOS) 16.0 SEM software from SPSS ,29, and Factor analysis was performed to explore the factor structure for the SIQ and SIQ-JR. Although the design of the SIQ/SIQ-JR was lack of theoretically based factor structure, Reynolds , the oriA short version of the SIQ-JR was explored for fast screening. The items kept in the short version were expected to have high correlations with the original scale. Corrected item-total correlations were ranked and a cutoff of 0.80 yielded four items with highest correlations as a short version, namely SIQ-JR-4 ,33. SpeaP > 0.05). However, significant gender differences were detected in both measurements: Female were more likely to report higher levels of suicidal ideation than male respondents .Out of 711 respondents, 44 reported suicidal ideation and 20 reported suicide attempts in the past 12 months. The prevalence rate (95% CI) was 6.2% 4.4-8.0%) and 2.8% (1.6-4.0%) respectively. The overall mean scores for the SIQ and SIQ-JR were 10.19 and 5.57 respectively. The distribution of both measurements’ scores was highly-skewed to the right , which was consistent with previous findings [.0% and 2The internal consistency of the SIQ/SIQ-JR was examined by employing Cronbach’s coefficient alpha, inter-item correlation, item-to-total scale correlation, and standard error of measurement (results not shown). Cronbach’s coefficients were found to be about 0.975 for the SIQ, and 0.951 for the SIQ-JR, in the overall, male, and female samples, which were similar in size to the findings reported by Reynolds . The homReynolds  proposed2/df > 10; root-mean-square error of approximation: RMSEA > 0.1; goodness-of-fit index: GFI < 0.9; and comptive fit index: CFI < 0.9). These results indicated that the proposed factorial structures of the SIQ/SIQ-JR by Reynolds might not be ideal. In fact, Reynolds’ factor analyses of the SIQ/SIQ-JR in the corresponding professional manual were exploratory, rather than confirmatory, since the questionnaires were not theoretically designed with distinct factors. Thus, a cautious claim was suggested by Reynolds that “use of these factors beyond simple descriptive purposes is not recommended at this time” [CFA was firstly carried out to test the goodness of fit of the one-factor and three-factor structures of the SIQ and the SIQ-JR respectively. Poor levels of goodness of fit were found in all models  among all 15 items in the SIQ-JR, namely item 2 (I thought about killing self), item 3 (I thought about how I would kill myself), item 4 (I thought about when I would kill myself), and item 11 (I wished I were dead). These items have covered specific suicidal wishes and plans, as major aspects along the continuum of suicidal ideation [A four-item short version of the SIQ-JR (SIQ-JR-4) was proposed by using the ranking of corrected item-total correlations, since this method can minimize the shared measurement error of item scores and total score . This SIP < 0.01 level. Moderate associations were found between the SIQ/SIQ-JR/SIQ-JR-4 and depression, hopelessness, and anxiety (r ranged 0.2-0.6). It is noticed that the SIQ-JR-4 was strongly correlated with the SIQ and SIQ-JR (r was 0.79 and 0.81 respectively). It is also noted that strength in correlations between the SIQ-JR-4 and depression, hopelessness, and anxiety was about the same as the correlations between the SIQ/SIQ-JR and the three psychological measurements.Spearman’s rank order correlations were calculated between the SIQ/SIQ-JR/SIQ-JR-4 and depression, hopelessness, and anxiety (results are displayed in Table P > 0.1) was detected among the AUCs of the SIQ, SIQ-JR, and SIQ-JR-4, yet they were all significantly (P < 0.05) larger than the AUCs of depression, hopelessness, and anxiety. Similarly, there was also no significant difference (P > 0.1) among the AUCs of the SIQ, SIQ-JR, and SIQ-JR-4 for screening suicide attempts in the past 12 months. The AUCs of the SIQ and SIQ-JR were significantly (P < 0.05) larger than those of depression, hopelessness, and anxiety. The AUC of the SIQ-JR-4 was also apparently (P < 0.05) larger than that of hopelessness and anxiety, though the difference of AUCs between the SIQ-JR-4 and depression did not reach the significant level of 0.05.ROC analyses were performed using the SIQ, SIQ-JR, and SIQ-JR-4 to predict respondents with suicidal ideation and suicide attempts in the past 12 months in comparison with the predicting effects of depression, hopelessness, and anxiety  performed less well, among which depression had the best predicting effects . These findings are very meaningful. They supported that the predicting and discriminative capacity of the SIQ-JR-4 was reasonably acceptable and hence the recommendation that it be a substitute of the SIQ/SIQ-JR for future quick assessment of suicidal ideation in young adolescents.This study presents the psychometric properties of the Chinese version of the SIQ and SIQ-JR in a Hong Kong community sample of adolescents. The findings supported that the Chinese SIQ and SIQ-JR have excellent internal reliability and concurrent validity. However, the potential factor structure of both measurements remained unclear. The factor structure proposed by the original author is not recommended for future direct application before further validation. The results also suggested that the four-item short version of the SIQ-JR is a useful alternative to the SIQ and SIQ-JR. It demonstrated good concurrent validity in identifying respondents with suicidal ideation (sensitivity = 0.95 and specificity = 0.71) and suicide attempts (sensitivity = 1 and specificity = 0.69) in the past 12 months. Efforts on shortening of psycho-social measurement scales should be recommended so as to reduce burden on respondents and to increase response rate .There are several limitations that should be carefully acknowledged. First, at least two concerns were raised due to the fact that the data employed for this study were a combined sample from two surveys. One issue is that the response rate of both surveys (61% and 62% respectively) was not very high. Yet, in consideration of the sensitive questions used, such response level was acceptable ,42. AnotDespite these limitations, there are many advantages for this study, including the community-based representative sample and a number of variables specifically designed to study suicidal ideation . A validated scale is highly needed for evaluating adolescent suicidality in Chinese-spoken societies. Additionally, this study provides a short form questionnaire as a convenient valuable tool for early screening of suicidal adolescents.SIQ: Reynolds’ Suicidal Ideation Questionnaire; SIQ-JR: Reynolds’ Suicidal Ideation Questionnaire-Junior; SIQ-JR-4: Items 2, 3, 4, and 11 of the SIQ-JR; CES-D: Center for epidemiologic studies depression scale; BHS: Beck’s hopelessness scale; DASS: Depression anxiety stress scale; SPSS: Statistical package for the social sciences; AMOS: Analysis of moment structures; EFA: Exploratory factor analysis; CFA: Confirmatory factor analysis; ROC: Receiver operating characteristics; AUC: Area under ROC curve; RMSEA: Root-mean-square error of approximation; GFI: Goodness-of-fit index; CFI: Comptive fit index; PPV: Positive predictive valueThe authors declare that they have no competing interests.YZ, PSFY and KF together designed the study. YZ analyzed the data and wrote the draft of the paper. All three authors have given final approval of this version to be published.


Work on protein structure prediction is very useful in biological research. To evaluate their accuracy, experimental protein structures or their derived data are used as the 'gold standard'. However, as proteins are dynamic molecular machines with structural flexibility such a standard may be unreliable.α of structures with identical sequences was relatively large, with the average of the maximum RMSD from each of the 137 sequences being 1.06 Å; (2) the derived data of the 3D structure was not constant, e.g. the highest ratio of the secondary structure wobble site was 60.69%, with the sequence alignments from structural comparisons of two proteins in the same family sometimes being completely different.To investigate the influence of the structure flexibility, we analysed 3,652 protein structures of 137 unique sequences from 24 protein families. The results showed that (1) the three-dimensional (3D) protein structures were not rigid: the root-mean-square deviation (RMSD) of the backbone CProteins may have several stable conformations and the data derived from resolved structures as a 'gold standard' should be optimized before being utilized as criteria to evaluate the prediction methods, e.g. sequence alignment from structural comparison. Helix/β-sheet transition exists in normal free proteins. The coil ratio of the 3D structure could affect its resolution as determined by X-ray crystallography. The best way to investigate the functions and mechanism of proteins at the molecular level is to obtain their three-dimensional 3D) structures -3. HowevD structuIn fact, thermodynamics and kinetics dictate that protein structures are not static . Work onhttp://www.rcsb.org/[α of two structures with identical sequences could reach 5.43Å. At the secondary structural level, we found helix/β-sheet transitions in normal free proteins which had only been reported previously in prion or protein complexes [In the present study, some redundant data deposited in PDB rcsb.org/ were colomplexes ,19-21. Fomplexes  that areCD-HIT  was util1. The sequential structures were determined by X-ray crystallography with resolution < 3.5Å;2. There were > 4 structures for each identical sequence;3. In each protein family, there were at least three unique proteins.In general, structures with resolution < 2.5 Å are considered reliable. However, analysis of structures with low resolution may supply some interesting information about protein flexibility. In the present study, 1,956 PDB entries were collected, with 1,588 having resolution < 2.5 Å . After that the ratio of the coil state under a certain resolution set was calculated for all structures. Then the Pearson's correlation coefficient (PCC) between the resolution and the ratio of coil state was calculated. Finally, a linear relationship between resolution and coil ratio was found. In addition, we checked the wobble ratio of structures with similar resolution.Here, we defined 'group pairs' as the results of the structural comparison of two structural groups. The 'group pairs' within the same family were utilized for sequence alignment variation analysis. If a site aligned the same residues in all sequence alignments from group pairs, it was defined as a 'common site'; or else defined as a 'multi-site'. If a site aligned a gap in all the comparisons, it was defined as a "gap site". We used equation 2 to calculate the ratio to reveal the sequence alignment variation.a Nis the average of the two proteins' length; x Nis the number of common sites (cN) or multi-sites (mN) or gap sites (gN); and x Ris the ratio of x Nto aN, c Rcorresponds to cN, m Rto mN, and g Rto gN.The sequence alignment based on structural comparison was not unique, so that we tried to optimize them. The secondary structure is usually used to help the sequence alignment and so we calculated the ratio of the secondary structure states of the three alignment states  for each family. For the wobble sites, if there were two secondary structure states in one site, then we added 0.5 to equivalent secondary structure state number. Finally, we calculated the average of these ratios.x Sand y Swhich contain n aligned sites [In theory, high structural similarity corresponds to low ED. RMSD and TM-score were utilized to measure the structural similarity. In each of the group pairs, two pairs of sequence alignments were selected based on the maximum and minimum of RMSD and TM-score. Equation 3 was utilized for ED calculation between the aligned sequences ed sites .th ialigned residues pairs in x Sand y Sfollowed the score matrix BLOSUM62. th isite pairs of x Sor the th isite pairs of yS, respectively. xSy) ED  are compared in Figure Except the impact of extrinsic factors, proteins are intrinsic not static, even when arrayed in a crystal . The proSecondary structure wobble is a result of structural flexibility. The maximum wobble site ratio was 60.69% Figure . Helix/βWe further found a strong linear relationship between the 'Wobble Total' and the maximum 'Wobble Single' Figure . The twoIn addition, the ratios of wobble sites in protein-protein complexes were higher than in free proteins . In addition, most of the original residues of these mutation sites were wobbles. Therefore, the sites in or around the functionally important regions should be of higher flexibility as noted by previous studies [Some structures contain few mutations, and we calculated the wobble sites ratio in these mutational sites and compared it to total sites Table . The mut studies .With decreasing resolution, the coil site ratio increased Figure . The anamax and m R. Therefore, with increasing structural difference, sequence alignment from structural comparison would be less reliable.There were 368 group pairs generated from structural comparisons among the structural groups in the same protein family. The sequence alignments from the structural comparison were not constant ; however, it was worse between maximum RMSD and its ED   1DSE: A and 2AS3: A, PF00141, 69Y, 70R; (B) 1AIG: M and 1PSS: M, PF00124, 26A, 27N; (C) 1GJM: A and 1T87: B, 82R, 83E, 86E, 87A. Besides these structure pairs, there are a total of nine families of helix/β-sheet transitions: PF00061, PF00124 and PF00139 are not enzymes; PF00067, PF00141 and PF00186 are enzymes with coenzymes and PF00215, PF00561 and PF01048 are enzymes without coenzymes.Click here for fileFigure S3: The wobble ratios of free proteins (A) and protein-protein complexes (B). Of the 137 structural groups, 64 were free proteins, and 73 contained protein-protein complexes.Click here for fileFigure S4: The wobble ratios of the structures without ligands (A) and with the same ligands (B). Of the 137 structural groups, 30 contained some structures without ligands, and 111 contained some structural pairs with the same ligands. Then their wobble ratios were counted.Click here for fileFigure S5: Resolution and wobble ratio. The structures were classified into six datasets based on their resolution value at a gradient value of 0.5 Å. Then the wobble ratio was calculated and the number of proteins in each dataset was marked on the histogram.Click here for fileFigure S6: TM-score and ED. The samples obeying the hypothesis are shown in blue diamonds, the exceptions are shown in red rectangles and where the ED difference was zero is shown in green triangles . (A) The distribution of the minimum/maximum TM-score. (B) The distribution of the ED of corresponding TM-score. (C) X-axis: differences of the maximum TM-score subtract the equivalent minimum TM-score; Y-axis: differences of the ED of the maximum TM-score subtract the ED of the minimum TM-score. (D) Gap difference of the exceptions. X-axis (gap-openings) and Y-axis (gap-extensions) are the differences of the gaps of the maximum TM-score subtract the minimum TM-score, respectively. The dots in quadrant IV indicate the gap-openings of the maximum TM-score are higher than the minimum TM-score, but the gap-extensions are reverse. The Venn diagram shows the dot number in quadrants I, II, III and IV, and on the axes and the origin e.g. there are 39 dots at the origin.Click here for fileFigure S7: RMSD and ED.Click here for file